Differential proteomic analysis and pathogenic effects of outer membrane vesicles derived from Acinetobacter baumannii under normoxia and hypoxia

Acinetobacter baumannii is a major causative agent of nosocomial infections and its outer membrane vesicles (AbOMVs) have been shown to be involved in pathogenicity by transporting virulence factors and transferring information for communication between pathogens and host cells. Despite the fact that the infected sites of A. baumannii such as lungs and skin soft tissues are hypoxic, most studies on AbOMV virulence have used AbOMVs prepared under aerobic conditions. The present study aims to elucidate the protein profile and pathogenic impact of AbOMVs released under hypoxic condition. AbOMVs were isolated from A. baumannii under normoxic and hypoxic conditions, and their protein profiles were compared. The different effects of both normoxic and hypoxic AbOMVs in cytokine response from mouse macrophages, cytotoxicity to the human lung epithelial cells, and bacterial invasion were then investigated. Our results showed that A. baumannii under hypoxia released larger amounts of OMVs with different protein profiles. Although the cytotoxic effect of AbOMVs from normoxia and hypoxia were comparable, AbOMVs from normoxia induced higher TNF-α production and invasion of Staphylococcus aureus and Pseudomonas aeruginosa than those from hypoxia. On the other hand, AbOMVs significantly enhanced A. baumannii invasion into lung epithelial cells in a dose-dependent manner. These results clearly demonstrate that AbOMVs released from normoxic and hypoxic have different impacts in pathogenesis. This finding provides new insight into the complex interactions between A. baumannii, coinfecting pathogens and host cells via OMVs, in particular the different pathogenic effects of AbOMVs under normoxic and hypoxic conditions.

Introduction Acinetobacter baumannii is an aerobic Gram-negative coccobacillus that is one of the causative agents of nosocomial infections [1]. It causes ventilator-associated pneumonia, blood stream infections, urinary tract infections, meningitis, and wound infections [2]. In recent years, the emergence of multidrug-resistant A. baumannii strains which are more difficult to treat than antibiotic-susceptible species has become one critical priority pathogen, and the World Health Organization is focusing on this issue [3][4][5].
Outer membrane vesicles (OMVs) released from Gram-negative bacteria have attracted attention as a virulence factor. The OMVs are generated by budding off from the outer membrane and are spherical in shape, 20-200 nm in diameter, surrounded by a lipid bilayer. These vesicles are composed of lipopolysaccharides (LPS), membrane proteins, lipids, cilia, cytosolic proteins, DNA, and RNA [6,7]. The ligands on the membrane surface can direct OMVs to bind multiple targets. Therefore, it has been suggested that the functions of OMVs include the transport of virulence factors such as outer membrane proteins, phospholipases and lipopolysaccharide, information transfer and communication between pathogens and host cells [8,9]. In addition, OMVs are considered to be stable vehicles because the enclosed structure of OMVs is able to protect the functional genes, DNA, RNA, proteins and other substances from degrading enzymes such as DNases, RNases and proteases [10].
The pathogenicity of A. baumannii during infectious process is associated with adhesion and invasion into the host cells, biofilm formation, induction of host cell death, and stimulation of host immune response [11,12]. A major virulence factor is outer membrane protein A (OmpA), which is associated with adhesion, invasion, and host cell apoptosis [13]. Since OMVs can deliver the virulence factors to host cells, there have been several reports on the presence of virulence factors including OmpA in AbOMVs and their pathogenicity such as host cell death and immune responses [14]. However, most studies on AbOMV virulence have used AbOMVs prepared from aerobic conditions [15], despite the fact that the infected sites such as lungs and skin soft tissues are hypoxic [16]. Importantly, there are no reports on the amount or pathogenicity of AbOMVs prepared from hypoxic conditions, even though other bacteria such as Pseudomonas aeruginosa has been shown to produce large amount of OMVs when cultured under stress conditions such as hypoxia [17,18].
The skin and lungs are the most common sites of A. baumannii infection in which other bacteria such as Staphylococcus aureus and P. aeruginosa often coexist. The polymicrobial infections by these pathogens are associated with severe prognosis of the diseases. One reason for this may be that the antibiotics are less effective. Carbapenem-resistant A. baumannii has been reported to protect other Gram-negative bacteria from exposure to β-lactam antibiotics [19]. It has also been reported that carbapenem-resistant A. baumannii enhanced carbapenem resistance in carbapenem-susceptible S. aureus [20]. Thus, these bacteria are thought to interact with each other and increase virulence as well as tolerance. However, the direct effect of AbOMVs on virulence of these coexisting pathogens has not been reported.
In this study, we investigated whether there is difference in the production and protein profiles of AbOMVs prepared under hypoxic condition (hAbOMVs) and under normoxic condition (nAbOMVs). In addition to the production and proteomic analysis, the pathogenic effects of hAbOMVs and nAbOMVs on host immune response, cytotoxicity, and A. baumannii invasion were compared. Moreover, the effects of these AbOMVs on the invasion of coexisting pathogens, S. aureus and P. aeruginosa, were also examined.

Bacterial strains and growth conditions
A. baumannii ATCC19606, S. aureus ATCC1718, and P. aeruginosa ATCC15692 were used in this study. They were cultured at 37˚C in tryptic soy broth (TSB; BD Bioscience, Sparks, MD). For purification of AbOMVs, A. baumannii was precultured for 16 h in TSB and used to inoculate into 4.8 L of Brain Heart Infusion medium (BD Bioscience) with 0.5% inoculum. The cultures were then placed under normoxic and hypoxic conditions for nAbOMVs and hAbOMVs, respectively. For normoxic condition, the cultures (400 mL medium in 1 L baffled flasks) were shaken at 125 rpm for 24 h, whereas for hypoxic condition, the cultures (400 mL medium in 500 mL normal conical flasks) were placed under static condition for 48 h. Thereafter, the supernatant was collected by centrifugation twice at 3,800 ×g, 4˚C for 60 min, filtrated through 0.45 μm filter (Nalgene Rapid-Flow Filters, Thermo Fisher Scientific, Waltham, MA) to remove remaining bacterial cells and kept -80˚C until AbOMVs purification. It should be noted that the oxygen concentration in uninoculated medium was measured using a JPB-70A dissolved oxygen analyzer (Shen Zhen Yage Technology, Guang Dong, China) [21]. The oxygen concentration for normoxia and hypoxia was 6.5 mg/L and 1.0 mg/L, respectively.
For bacterial infection assay, A. baumannii, S. aureus, and P. aeruginosa were precultured for 16 h and inoculated into 20 mL of TSB. The cultures were incubated at 37˚C under aerobic condition by shaking at 125 rpm for 5 h. The bacterial cells were collected, washed twice with phosphate-buffered saline (PBS), and adjusted to an appropriate concentration by calculating via conversion of optical density at 600 nm.

Purification of AbOMVs
AbOMVs in the culture supernatant of A. baumannii were harvested by ultracentrifugation at 128,400 ×g, 4˚C for 90 min using a Himac CP80NX Preparative Ultracentrifuge (HITACHI, Tokyo, Japan). After removal of the supernatant, crude pellet containing AbOMVs was then washed and suspended in 2 mL of PBS. AbOMVs were further purified using an OptiPrep™ density gradient according to the standard protocol [22] with some modifications. Briefly, a discontinuous iodixanol gradient [45%, 35%, 30%, 25% and 15% (w/v) iodixanol solutions (OptiPrep™; Sigma Aldrich, St. Louis, MO) in 0.25 M sucrose/10 mM Tris, pH 7.5] was prepared. A 320 μL volume of crude-AbOMVs suspension was overlaid on the discontinuous iodixanol gradient and ultracentrifuged for 16 h at 100,300 ×g, 4˚C. Thereafter, six fractions (720 μL each) were collected from the top of the gradient. Then, each fraction was diluted in PBS, and again harvested by ultracentrifugation at 99,800 ×g, 4˚C for 3 h. The pellet was washed once and resuspended in an appropriate volume of PBS. Protein concentration of each fraction was measured by Bradford protein assay using Bio-Rad Protein Dye Reagent Concentrate (Bio-Rad Laboratories, Inc., Hercules, CA). The AbOMVs containing fraction was analyzed under transmission electron microscope (JEM-1230, JEOL, Tokyo, Japan) with negative staining (TI blue, Nisshin EM Co. Ltd, Tokyo, Japan). The particle concentration and size distribution of AbOMVs were analyzed using qNano instrument (Izon Science, Oxford, United Kingdom) according to the manufacture's instruction. Briefly, the purified AbOMVs were diluted in Measurement Electrolytes (qNano reagent kit) containing 0.03% Tween 20 (FUJIFILM Wako Pure Chemical Corporation) and analyzed using NP200 nanopore membranes in comparison with 200-nm calibration particles by running at 0.62 V. Izon Control Suite software version 3.3.2.2001 was used for data analysis.

Proteomic analysis of AbOMVs
Quantitative proteomic analyses of nAbOMVs and hAbOMVs were performed in triplicate by liquid chromatography-tandem mass spectrometry as described previously [23]. Briefly, the acetone-precipitated proteins of AbOMVs were denatured with 50% trifluoroethanol and reduced with 4 mM dithiothreitol. Free cysteine residues were alkylated prior to trypsinization. The peptides were desalted and separated using liquid chromatography. The mass spectrometer was set in an information-dependent acquisition mode. Acquired spectra were searched against the A. baumannii ATCC19606 proteins (NCBI Genbank: CP045110.1). Positive identification was considered when identified proteins and peptides reached a 1% local false discovery rate. The proteomic data obtained in this study are available using accession number PXD037014 and JPST001865 for Proteome Xchang and jPOST Repository, respectively. Differential proteomics between nAbOMVs and hAbOMVs was then analyzed. Compute pI/Mw tool-ExPASy (https:// web.expasy.org/compute_pi/), PsortB v.3.0 (https://www.psort.org/psortb/) were used for predicting molecular weight and subcellular localization of each identified protein, respectively.

Cytokine assay
RAW264.7 cells (1 × 10 6 cells/mL/well) were seeded in 24-well culture plates and stimulated with 12.5 μg/mL AbOMVs for 48 h. The culture supernatant was then collected by centrifugation twice at 5,000 ×g, 4˚C for 10 min to remove the residual cells. The titers of TNF-α, IL-6, and IL-10 were measured using Mouse ELISA kits (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

Cytotoxic effect of AbOMVs
A549 cells (1 × 10 4 cells/100 μL/well) were seeded in 96-well plates and incubated with 0, 1, 5, 10, 25 and 50 μg/mL AbOMVs at 37˚C under 5% CO 2 . At 12 h of incubation, A549 cells were washed once with Hanks' Balanced Salt Solution (HANKS; Nissui Pharmaceutical Co.) to remove AbOMVs. Cells were then maintained in 100 μL medium prior to cell viability assay. WST-1 Cell Proliferation Reagent (Sigma-Aldrich) was used for determining cell viability was determined using according to the manufacturer's instruction. Briefly, WST-1 reagent (10 μL) was added into each well directly and incubated at 37˚C under 5% CO 2 . After color development, the optical density at 450 nm was measured using a microplate reader (MULTISKAN Sky, Thermo Fisher Scientific). The absorbance values were subtracted with background controls (medium without A549 cells containing 0, 1, 5, 10, 25 and 50 μg/mL AbOMVs, respectively). The viability of A549 cells without AbOMVs is referred to 100%.

Bacterial infection assay
A549 cells (5 × 10 4 cells/mL/well) were seeded in 24-well plates and incubated at 37˚C, under 5% CO 2 . At 48 h after incubation, A549 cells were washed and infected with A. baumannii, S. aureus, or P. aeruginosa at MOI = 50 in the presence of AbOMVs. The infection time was 5 h for A. baumannii, and 2 h for S. aureus and P. aeruginosa. Afterwards, the bacterial cells were removed, and the cells were washed twice with HANKS solution and once with PBS. To eliminate all extracellular A. baumannii and S. aureus, the A549 cells were treated with 0.2% Lysostaphin (FUJIFILM Wako Pure Chemical Corporation). On the other hand, the A549 cells were treated with 120 μg/mL of gentamicin (Wako Pure Chemical Corporation) to eliminate the extracellular P. aeruginosa. At 1 h of antibiotic treatment, the cells were then washed with HANKS solution and once with PBS again. To enumerate the invading A. baumannii, the A549 cells were lysed with 0.2% (3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS; DOJINDO, Kumamoto, Japan) for 1 h. To enumerate the invading S. aureus and P. aeruginosa, the A549 cells were lysed with 1% CHAPS for 15 min. Then, the bacterial cell suspensions were diluted and plated on tryptic soy agars. The colonies were counted at 24 h after incubation.

Statistical analysis
Statistical differences were analyzed using the method mentioned in each figure legend. A Pvalue less than 0.05 is considered statistically significant.

Purification of nAbOMVs and hAbOMVs
To prepare nAbOMVs and hAbOMVs, A. baumannii was cultured under aerobic and static condition, respectively. The growth of A. baumannii under aerobic condition reached an OD 600nm of about 2.0 after 24 h of incubation, and that under static condition reached an OD 600nm of about 1.0 after 48 h. The culture supernatants from both conditions were then collected and subsequently purified by step-gradient ultracentrifugation. From six separated fractions (F1 to F6), the purified AbOMVs from each culture condition were detected in F2 as confirmed by negative-staining transmission electron microscopy (Fig 1A and 1B). The particles with an enclosed structure of approximately 200 nm in diameter were observed. Nanoparticle tracking analysis revealed that the average particle sizes of nAbOMVs and hAbOMVs were very similar, with 187 nm and 189 nm, respectively (Table 1). From 1 μg of protein, the number of OMV particles from normoxia and hypoxia was slightly different with 2.71 × 10 8 and 2.19 × 10 8 particles, respectively. Obviously, from an equivalent volume of culture supernatant (4.8 L), the protein yield and total particle number of hAbOMVs were approximately 3 times higher than those of nAbOMVs (Table 1 and S1 Dataset).

Differential proteomic analysis between nAbOMVs and hAbOMVs
To investigate the differences in protein profiles between nAbOMVs and hAbOMVs, quantitative proteome analysis was performed. A total of 584 proteins were identified from the triplicated preparations of nAbOMVs and hAbOMVs. The differential comparisons between nAbOMVs and hAbOMVs indicated that 30 proteins were significantly higher in nAbOMVs (P < 0.05) ( Table 2). Among these, six proteins are hypothetical proteins with unknown function. Alkyl hydroperoxide reductase C was detected only in nAbOMVs. The proteins enriched more than 10-fold in nAbOMVs were polysaccharide biosynthesis tyrosine autokinase, dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex, outer-membrane lipoprotein carrier protein, and polysaccharide biosynthesis export family protein. The proportion of cytosolic, cytoplasmic membrane, periplasmic. outer membrane, extracellular and unknown location proteins was 7:6:3:2:1:11. The proteins that have been shown to associate with A. baumannii virulence were outer membrane receptor FepA, polysaccharide biosynthesis tyrosine autokinase and taurine ABC transporter substrate-binding protein [24].
The proteins enriched in hAbOMVs with P value < 0.05 are shown in Table 3. Of these 25 proteins, two are hypothetical proteins with unknown function and several proteins are enzymes involving in metabolism. GntR family transcriptional regulator was detected only in hAbOMVs. The proteins enriched more than 10-fold in hAbOMVs were haloacid dehalogenase (HAD) hydrolase, family IB and methionine aminopeptidase. The proportion of cytosolic, cytoplasmic membrane, periplasmic. outer membrane, extracellular and unknown location proteins was 14:5:0:0:0:6. Among these proteins, outer-membrane lipoprotein LolB, and highaffinity zinc transporter ATPase are lipoproteins that have been shown to associate with A. baumannii virulence [24].
Since OmpA is a key virulence factor of A. baumannii, the amount of OmpA in nAbOMVs and hAbOMVs was also focused. As shown in Table 3, the amount of OmpA was 2.2-fold higher in hAbOMVs than that in nAbOMVs, but the statistical analysis shows no significant difference (P = 0.252).
These proteomic data demonstrated that the types and amounts of several proteins involved in virulence of A. baumannii differed between nAbOMVs and hAbOMVs. Therefore, we further examined the difference in the effects of nAbOMVs and hAbOMVs on host immune response, cytotoxicity, and bacterial infections.

Effects of nAbOMVs and hAbOMVs on host immune responses
To examine the different effects of nAbOMVs and hAbOMVs on host immune responses, mouse macrophage RAW264.7 cells were prepared and stimulated with 12.5 μg/mL of each AbOMVs. At 48 h of stimulation, cytokines in the culture supernatant were measured by ELI-SAs. As shown in Fig 2, the purified AbOMVs from both normoxia and hypoxia significantly Total protein (mg) ± SD Particle diameter (nm) ± SD Particle number (×10 8 per 1 μg of protein) ± SD Total particle number (×10 11  stimulated the production of TNF-α, IL-6, and IL-10 from RAW264.7 cells compared to non-AbOMV control. These results suggest that AbOMVs from both conditions strongly stimulate host immune responses. In addition, TNF-α production by nAbOMV stimulation was significantly higher than that by hAbOMVs (Fig 2A and S2 Dataset). However, this different effect was not observed in IL-6 and IL-10 production (Fig 2B and 2C, and S2 Dataset).

Cytotoxic effects of nAbOMVs and hAbOMVs to human lung epithelial cells
The cytotoxic effects of nAbOMVs and hAbOMVs to A549 lung epithelial cells were examined. At 12 h after incubation of A549 cells with AbOMVs, WST-1 reagent was added, and the absorbance at 450 nm was measured. The cytotoxic effects of nAbOMVs and hAbOMVs to  Fig 3 and S3 Dataset. In the presence of AbOMVs, the cell viability was decreased compared to non-AbOMV control. The decrease in cell viability was dosedependent up to a concentration of 10 μg/mL. At AbOMVs concentrations above 10 μg/mL, the cell viability remained constant at approximately 63%. nAbOMVs and hAbOMVs reduced cell viability with a similar trend with no significant difference.  The effects of AbOMVs on infections of the common coexisting pathogens, S. aureus and P. aeruginosa, were also investigated. A549 cells were infected with S. aureus or P. aeruginosa (MOI = 50) in the presence of 12.5 mg/mL of nAbOMVs or hAbOMVs for 2 h, and the invading bacterial cells were then enumerated. The results are expressed as relative numbers to the invading S. aureus or P. aeruginosa without AbOMVs (set at 1.0). As shown in Fig 4B and S4 Dataset, nAbOMVs promoted the invasion of both S. aureus and P. aeruginosa into A549 cells compared to non-AbOMV control (referred to 1.0). On the other hand, hAbOMVs enhanced the invading number of S. aureus but not P. aeruginosa. Overall infection results showed that nAbOMVs significantly promoted invasion of S. aureus and P. aeruginosa more than hAbOMVs. This was inconsistent with their effects on A. baumannii infection (Fig 4A).

Discussion
A. baumannii is a major causative agent of nosocomial infections [2]. At infected sites such as skin and lungs, this microorganism often co-infects with S. aureus and P. aeruginosa, which results in severe prognosis [25,26]. It has been known that the extracellular membrane vesicles released from bacteria play an important role in bacterial pathogenicity. Based on structure and composition, these vesicles can deliver the virulence factors directly to the host cells and  At 12 h of incubation, 10 μL of WST-1 reagent was added into each well and the absorbance was measured at 450 nm. Cell viability in the reactions without AbOMVs (control) was calculated as 100% (n = 7 from 3 independent experiments). P-value was calculated using Kruskal Wallis H-test with post hoc Mann-Whitney U-test. Asterisks indicates significant difference to non-AbOMV control ( � : P < 0.05, �� : P < 0.01). P-value of difference between nAbOMVs and hAbOMVs was calculated using Mann-Whitney U-test. ns: not significant.  Bacterial number from the condition without AbOMVs is referred to 1.0 (dotted line). (B) A549 cells were infected with S. aureus or P. aeruginosa at MOI = 50 in the presence of 12.5 μg/mL AbOMVs for 2 h (n = 6 from 2 independent experiments). Invasion efficacy was expressed as the relative number to the condition without AbOMVs, which is referred to 1.0 (dotted line). P-value of difference to non-AbOMV control was calculated using Kruskal Wallis H-test with post hoc Mann-Whitney U-test. � : P < 0.05, �� : P < 0.01. P-value of difference between nAbOMVs and hAbOMVs was calculated using Mann-Whitney U-test. †: P < 0.05, ‡: P < 0.01.
https://doi.org/10.1371/journal.pone.0283109.g004 transmit signal information to other pathogens for promoting virulence [14,27]. The composition and virulence of AbOMVs prepared under aerobic condition, which is suitable for A. baumannii growth, have been reported [28]. However, it has been reported that infected tissues are hypoxic because of increased oxygen consumption due to activation of immunerelated cells and decreased oxygen supply due to damage to blood vessels. Thus, while normal lung tissue is in contact with the atmosphere and have a high oxygen concentration, the infected area is thought to be hypoxia [16]. Under this hypoxic stress, bacteria need to alter their gene expression profiles to survive, especially the proteins related to nutrient transport and metabolism [29]. It has been demonstrated that the amount of OMVs released from P. aeruginosa is altered under hypoxia [17]. Therefore, we hypothesized that the amount and protein profile of AbOMVs prepared under hypoxia would differ from those under normoxia. We also expected that the difference in the protein profiles in the AbOMVs would alter their role in virulence. Therefore, in the present study, AbOMVs under normoxic and hypoxic conditions were purified and their different effects on host immune response, cytotoxicity and bacterial infections were investigated.
As shown in Table 1, there was no significant difference in the particle size and particle concentration (per 1 μg protein) between the purified nAbOMVs and hAbOMVs. These particle sizes were comparable to that of A. baumannii OMVs isolated by Sun Li, et al. [15]. However, the protein yield as well as the total particle number of hAbOMVs were approximately 3-fold higher than those of nAbOMVs. These obtaining yields were constant from triplicated preparations. Together with the differences in growth of A. baumannii under normoxic and hypoxic conditions, our results indicate that the hypoxia, a stress condition for A. baumannii, remarkably promotes AbOMVs production over normoxic condition.
Differential proteomic analysis was performed to observe differences in the protein profiles of nAbOMVs and hAbOMVs. As shown in Tables 2 and 3, 30 proteins were significantly enriched in nAbOMVs and 25 proteins were significantly enriched in hAbOMVs. The enriched proteins in nAbOMVs that have been shown to play a role in A. baumannii virulence are the outer membrane receptor FepA (TonB-dependent siderophore receptor), polysaccharide biosynthesis tyrosine autokinase and taurine ABC transporter substrate-binding protein.
FepA has been shown to be involved in iron-scavenging system and promote the growth of A. baumannii [30,31]. The tyrosine autokinase is markly elevated to export the exopolysaccharide during biofilm formation [32], and the taurine ABC transporter substrate-binding protein is involved in the transport of taurine as a sulfur source and in competition for taurine utilization between the nosocomial pathogens and host [33].
Proteins enriched in hAbOMVs that have been shown to be involved in the virulence of A. baumannii include outer-membrane lipoprotein LolB and high-affinity zinc transporter ATPase. The outer-membrane lipoprotein LolB constitutes the Lol lipoprotein transport system and transports lipoproteins to the outer membrane in especially LPS-deficient A. baumannii [34]. The high-affinity zinc transporter ATPase ZnuC constitutes the ZnuABC transporter to acquire zinc within the hostile environment of the host [35].
In addition to these known proteins, the hypothetical proteins with unknown function might also contribute to the virulence of AbOMVs. Moreover, OMVs from Gram-negative bacteria are known to carry LPS, peptidoglycan as well as lipoproteins to induce inflammatory response via pattern recognition receptor-mediated interactions [36,37]. Therefore, we further compared the cytokine responses from mouse macrophages after nAbOMVs and hAbOMVs stimulations. As shown in Fig 2, the production of TNF-α, IL-6 and IL-10 from RAW264.7 cells was significantly increased after stimulations with nAbOMVs and hAbOMVs. Although there was no significant difference in the production of IL-6 and IL-10 between nAbOMVs and hAbOMVs stimulation, the TNF-α production caused by nAbOMVs stimulation was significantly higher than that by hAbOMVs. A previous study has suggested that OmpA in OMVs is a key factor for cytokine stimulation because the OMVs from ΔOmpA mutant of A. baumannii induce lower levels of TNF-α and IL-6 compared to OMVs of the wild-type strain [14]. Although our proteomic analysis revealed that OmpA seems to be enriched in hAbOMVs for (2.20-fold change), there was no statistically significant difference (P value = 0.252). In contrast, the proportion of polysaccharide biosynthesis-related proteins and the periplasmic proteins was enriched in nAbOMVs. Thus, it is possible that polysaccharides and peptidoglycans in periplasm may be enriched in nAbOMVs, and these molecules may contribute to the high titer of TNF-α due to nAbOMVs stimulation.
It has been shown that AbOMVs exhibit the cytotoxic effect to the host cells [38], and a key toxic factor is OmpA because Skerniškytė et al. demonstrated that the cytotoxicity of AbOMVs from A. baumannii lacking OmpA decreased [14]. Since our proteomic analysis revealed that the amount of OmpA in nAbOMVs and hAbOMVs was not significantly different (P value = 0.252), it was not surprising that the cytotoxic effect of nAbOMVs and hAbOMVs to human lung epithelial cells was comparable (Fig 3). Interestingly, the cytotoxic effect of both AbOMVs was not increased at the concentration above 10 μg/mL. Further experiments are required to explain the details of this mechanism.
The effect of nAbOMVs and hAbOMVs on bacterial infection was also examined to determine whether nAbOMVs and hAbOMVs promote A. baumannii invasion. It has been shown that OmpA is required for A. baumannii to adhere to and invade the epithelial cells [12]. The number of ΔompA A. baumannii infections into epithelial cells significantly decreased compared to the wild type, and pretreatment of recombinant OmpA to the host cells inhibits the interaction of A. baumannii with the epithelial cells [12]. Since OmpA was comparably detected in both nAbOMVs and hAbOMVs, we expected that both AbOMVs may inhibit A. baumannii internalization. Surprisingly, our results showed that nAbOMVs did not inhibit A. baumannii invasion even the concentration was up to 50 μg/mL. In contrast, hAbOMVs significantly enhanced A. baumannii invasiveness in a dose-dependent manner (Fig 4A). These results suggest that some proteins enriched in hAbOMVs (in Table 3) may act as effectors to regulate cytoskeleton dynamics and induce A. baumannii uptake into lung epithelial cells. In particular, under stressful hypoxic condition, A. baumannii may highly release these effectors together with OMVs to promote its infectivity.
A. baumannii often causes infections of the skin and lungs where P. aeruginosa and S. aureus usually coexist. We hypothesized that there may be AbOMV-mediated interactions between these bacteria. Therefore, the effect of AbOMVs on S. aureus and P. aeruginosa infection was also examined. In contrast to the effects of AbOMVs on A. baumannii infection, nAbOMVs promoted S. aureus and P. aeruginosa invasion into A549 epithelial cells. Although hAbOMVs also promoted S. aureus invasion, these vesicles did not affect P. aeruginosa invasiveness. The invasion mechanism of S. aureus into the epithelial cells is suggested to be mediated by fibronectin that forms bridging between host cell and S. aureus surface [39]. In addition, the invasion mechanism of P. aeruginosa is mediated by the formation of bacterial aggregates on the surface of epithelial cells [40]. Since these mechanisms are different from that of A. baumannii which invades into the epithelial cells via a zipper-like mechanism [12,41], the different effectors in nAbOMVs and hAbOMVs may reflect their different effects on these bacterial infections. During infection, A. baumannii probably competes other bacterial species [42]. In particular, under stress of hypoxic condition, A. baumannii may have highly competitive with P. aeruginosa because both of them preferentially use aerobic respiration [43,44]. Therefore, a high hAbOMVs production with the effective molecules from hypoxic condition would be a beneficial strategy for A. baumannii to promote its own survival and outcompete P. aeruginosa. It is important to noted that OMVs do not contain only proteins or lipoproteins. The other bioactive molecules such as LPS, peptidoglycan, DNA and RNA in OMVs are also possible to contribute to these effects. Although further investigation of the molecular mechanisms of AbOMVs involved in bacterial infection into the host cells is required, we expected that the proteins reported in this study would provide great benefits for progression in the research field of OMVs and their relationship to infection. Taken all together, our results indicated AbOMVs from normoxia and hypoxia exhibited different effects to promote the infections of A. baumannii and other bacteria.

Conclusion
This study demonstrated that A. baumannii under hypoxic condition produces higher amount of OMVs than that under normoxic condition. The protein profile and the pathogenic effect of AbOMVs under normoxic and hypoxic are also different. Although the cytotoxic effect of nAbOMVs and hAbOMVs to human lung epithelial cells was comparable, the OMVs released from A. baumannii under normoxia promoted higher TNF-α production and enhanced S. aureus and P. aeruginosa internalization into lung epithelial cells. On the other hand, OMVs release from A. baumannii under hypoxia promoted invasion of A. baumannii into lung epithelial cells. This finding provides new insight into the complex interactions between infecting pathogens and host mediated by OMVs, in particular the different effect under normoxic and hypoxic conditions. Supporting information S1 Dataset. Total protein, particle diameter and particle number of nAbOMVs and hAbOMVs obtained from 4.8 L of A. baumannii ATCC19606 culture supernatants. Validation: Sachio Suzuki.